Background: Hypothyroidism is associated with dysfunction of the bone turnover with reduced osteoblastic bone formation and osteoclastic bone resorption. Mesenchymal stem cells (MSCs) secrete various factors and cytokines that may stimulate bone regeneration. The aim of this study was to determine the effects of MSCs-conditioned medium (CM) in hypothyroidism male rats after inducing bone defect. Methods: In this study, 24 male rats were randomly assigned to three groups: (I) hypothyroidism + bone defect (HYPO), (II) hypothyroidism + bone defect + CM (HYPO + CM), and (III) no hypothyroidism + bone defect (control). Four weeks after surgery, the right tibia was removed, and immediately, biomechanical and histological examinations were performed. Results: The results showed a significant reduction in bending stiffness (32.64 ± 3.99), maximum force (14.63 ± 1.89), high stress load (7.59 ± 2.31), and energy absorption (12.68 ± 2.12) at the osteotomy site in hypothyroidism rats in comparison to the control and hypothyroidism + condition medium groups (p < 0.05). There was also a significant decrease in the trabecular bone volume (3.86 ± 3.88) and the number of osteocytes (5800 ± 859.8) at the osteotomy site in hypothyroidism rats compared to the control and hypothyroidism + condition medium groups (p < 0.01 and p < 0.02, respectively). Conclusion: The present study suggests that the use of the CM can improve the
fracture regeneration and accelerates bone healing at the osteotomy site in hypothyroidism rats.